Electrochemically Synthesized Polyacrylamide Gel and Core–Shell Nanoparticles for 3D Cell Culture Formation

Biocompatible polyacrylamide gel and core–shell nanoparticles (NPs) were synthesized using a one-step electrochemically initiated gelation. Constant-potential electrochemical decomposing of ammonium persulfate initiated the copolymerization of N-isopropyl acrylamide, methacrylic acid, and N,N′-methylenebisacrylamide monomers. This decomposing potential and monomers’ concentrations were optimized to prepare gel NPs and thin gel film-grafted core–shell NPs. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging confirmed the gel NP formation. The lyophilized gel NPs and core–shell NPs were applied to support the three-dimensional (3D) cell culture. In all, core–shell NPs provided superior support for complex 3D tissue structures.


S1. Effect of initiator concentration
A sufficient amount of peroxydisulfate initiator ions in the aqueous solution for polymerization is necessary to generate a sufficiently high number of active centers that could propagate to undergo a phase transition to form precursor particles or short oligomer chains. Monomers polymerized on these nuclei centers forming soft colloidal gel particles. Therefore, the amount of initiator in the solution was varied. A sufficient number of free radicals was generated in the solution for gel particle preparation at the initiator concentration as low as 15 mM.

S2. Effect of polymerization quencher
This radical polymerization might not be sufficiently quenched after seizing the electrochemical reaction.
Therefore, a polymerization inhibitor, vis., hydroquinone monomethyl ether, was added after stopping electroinitiation. Three different inhibitor concentrations were used, i.e., 5, 15, and 25 mM, to study the polymerization chain inhibition effect on the gel particles' morphology ( Figure S3). Nevertheless, globular gel particles of different sizes were obtained in each case.

S3. The process in the vicinity of the electrode surface
Electrochemically initiated N-isopropylacrylamide, methacrylic acid, and N,N'methylenebisacrylamide polymerization is started by dissociating persulfate at the suitable potential. A pathway of persulfate reduction is proposed below. This pathway involves a direct electron transfer to the anion in the solution.
Similarly, as previously described, 1,2 with time, more and more ammonium persulfate is cleaved at the potential of -0.60 V (Equation S1a), and the amount of water-soluble free radicals

S4. MTT assay for IC 50 value
The cell solution after the passage was diluted to an approximate number of ~10 000 cells/well (controlled with Countess II Cell Counter) and seeded into a 96-well plate (Greiner Bio-One).
Then, cells were incubated for 24 h at 37 C to enable cell attachment. The medium was removed, and NIPAM-MA-BIS gels were added to the fresh culture medium at different concentrations (1000 μg/mL to 2 μg/mL). We performed five repeats for each concentration.
The absorbance in each well was measured at 540 nm using a Synergy HTX multi-mode reader (BioTek). Two independent cytotoxicity assays were performed.

Data analysis
The percentage of viable cells in each sample compared to the control was calculated from the absorbance measurement data using Equation S3: where A1 and A2 were upper and lower asymptotes, respectively; the IC50 values were fitting parameters of the model. The fitting results are presented in Figures S6 and S7. The results obtained enabled determining the IC50 value as 485 μg/mL. The dose-response graph is shown in Figure S6 in the Supporting Information. The IC50 value corresponds to 15-fold coverage of the growth surface ( Figure S7 in the Supporting Information). A significant decrease in cell S-10 viability was observed for the highest concentrations (33% of living cells for 1 000 μg/ml).
However, it is essential to remember that the IC50 value is closely related to the nanoparticles' coverage of the surface area available for cell growth.